High affinity synbodies for influenza

ABSTRACT

Composition of synbodies that bind influenza. The synbodies are composed of two peptides joined on a scaffold.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under W911 NF-10-1-0299awarded by the US Army Research Office. The US government has certainrights in the invention.

FIELD OF THE INVENTION

This invention is directed to a composition of synbodies that bindinfluenza, more particularly, the synbodies are composed of two peptidesjoined on a scaffold.

BACKGROUND OF THE INVENTION

Influenza is a critical public health concern and each year 3,000-52,000people die from influenza infection the United States. Influenza is aconstantly evolving pathogen that has rapidly developed resistance tocurrently approved antivirals. Rapid diagnosis and typing of influenzainfection plays a vital role in surveillance and use of antiviraltherapeutics. Recently, several ELISA kits that use antibodies forinfluenza detection have been approved for use in point-of-carefacilities but there have been concerns with the variability,sensitivity, selectivity and stability of these tests, illustrating theneed for additional influenza affinity ligands. Ideally, these ligandscould be used in diagnostic and therapeutic applications and should berapidly produced to keep pace with viral antigenic drift and shift.International Publication No. WO08/048970, published Apr. 24, 2008, andentitled “Synthetic Antibodies” describes methods for isolating a classof molecules termed synthetic antibodies or synbodies. Synbodies containat least two compounds, such as short peptides, joined via a linker.Although the affinity of individual compounds for a target is typicallyweak, the combination of compounds can bind desired target withaffinities comparable to antibodies. Synbodies have advantages overantibodies resulting in part from their smaller size. These advantagesmay include ease of initial isolation, ease and cost of production, andimproved tissue penetration. Synbodies, can be developed by linking twolow affinity 15-20 amino acid (aa) long peptides to produce a highaffinity synbody for a target protein or bacteria.

In general, synbodies comprising affinity elements and linkers that canbe synthesized by standard solid phase synthesis techniques can besynthesized either by addition of amino acids or other monomers in astepwise fashion, or by joining preassembled affinity elements andlinkers or other presynthesized subunits. Techniques for stepwisesynthesis of peptides and other heteropolymers are described by e.g.,Atherton E, Sheppard RC: Solid Phase peptide synthesis: a practicalapproach. Oxford, England: IRL Press; 1989, and Stewart J M, Young J D:Solid Phase Peptide Synthesis, 2d Ed. Rockford: Pierce Chemical Company;1984, which are incorporated herein by reference. Examples ofconjugation chemistries have been discussed in International PatentApplication Publication Nos. WO08/048970 and WO/2009/140039, publishedNov. 19, 2009. The use of “click” chemistry to perform conjugationsbetween biopolymers and other heteropolymers is also described in Kolbet al., Angewandte Chemie-International Edition 2001, 40(11):2004 andEvans, Australian Journal of Chemistry 2007, 60(6):384-395, which areincorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one embodiment, an agent comprises a first peptide having an aminoacid sequence comprising a first mutant of SEQ. ID NO: 1 and a secondmutant of SEQ ID NO: 1, wherein the first and second mutants are linkedand consist of linked mutant peptides SEQ ID NOS: 2-2, 3-3,5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15.

In another embodiment, the first and second mutant peptides are linkedto a scaffold structure to synthesize a composition having an affinityfor a target molecule.

In another embodiment, the synthesized composition has an affinity forinfluenza viruses.

In another embodiment, the scaffold structure has the structure:

In another embodiment, the scaffold structure has the structure:

In another embodiment, a composition comprises or consists of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In another embodiment, a composition comprises or consists of thestructure

where In5 comprises or consists of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15.

In another aspect, a method of detecting an influenza virus, comprisescontacting a sample suspected of containing influenza virus with anagent of claim 1, and measuring binding of the agent to the samplecompared with a control lacking influenza virus, an increase in bindingrelative to the control providing an indication of presence of influenzavirus.

In another aspect, the sample is from a patient.

In another aspect, the agent further comprises a therapeutic moleculelinked to the agent.

In another aspect, a method of diagnosing a patient for an influenzavirus, comprises contacting a sample suspected of containing influenzavirus with a synthesized agent, and measuring binding of the agent tothe sample compared with a control lacking influenza virus, an increasein binding relative to the control providing an indication of presenceof influenza virus.

In another aspect, the agent further comprises a therapeutic moleculelinked to the agent.

In another aspect, a method of detecting an influenza virus, comprisescontacting a sample suspected of containing influenza virus with acomposition having a synthesized agent and scaffold structure, andmeasuring binding of the composition to the sample compared with acontrol lacking influenza virus, an increase in binding relative to thecontrol providing an indication of presence of influenza virus.

In another aspect, the sample is from a patient.

In another aspect, the agent further comprises a therapeutic moleculelinked to the agent.

In another aspect, a method of diagnosing a patient for an influenzavirus, comprising contacting a sample suspected of containing influenzavirus with a synthetic composition comprising In5, and measuring bindingof the composition to the sample compared with a control lackinginfluenza virus, an increase in binding relative to the controlproviding an indication of presence of influenza virus.

In another aspect, the agent further comprises a therapeutic moleculelinked to the agent.

In another aspect, a diagnostic agent for influenza comprising a firstpeptide having an amino acid sequence comprises a first mutant of SEQ.ID NO: 1 and a second mutant of SEQ ID NO: 1, wherein the first andsecond mutants are linked and consist of linked mutant peptides SEQ IDNOs: 2-2, 3-3, 5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15;

wherein the first and second mutant peptides are linked to a scaffoldstructure to synthesize a composition having an affinity for influenzaviruses; and

wherein contacting a sample suspected of containing influenza virus withthe synthesized composition, and measuring binding of the synthesizedcomposition to the sample compared with a control lacking influenzavirus, an increase in binding relative to the control providing anindication of presence of influenza virus.

In yet another aspect, a therapeutic agent for influenza comprises afirst peptide having an amino acid sequence comprising a first mutant ofSEQ. ID NO: 1 and a second mutant of SEQ ID NO: 1, wherein the first andsecond mutants are linked and consist of linked mutant peptides SEQ IDNOs: 2-2, 3-3, 5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15;

wherein the first and second mutant peptides are linked to a scaffoldstructure to synthesize a composition having an affinity for influenzaviruses; and

wherein the agent further comprises a therapeutic molecule linked to thesynthesized composition.

BRIEF DESCRIPTION OF THE DRAWINGS

While the novel features of the invention are set forth withparticularity in the appended claims, the invention, both as toorganization and content, will be better understood and appreciated,along with other objects and features thereof, from the followingdetailed description taken in conjunction with the drawings, in which:

FIG. 1 shows an overview of an example of high affinity synbodydevelopment process.

FIG. 2A shows a structure of a first scaffold used to constructinfluenza binding synbodies.

FIG. 2B shows a structure of a second scaffold used to constructinfluenza binding synbodies.

FIG. 3A and FIG. 3B show binding curves of biotin-labeled peptidestested against Influenza coated ELISA plates where peptide concentrationis plotted versus A450 (y-axis).

FIG. 4 shows binding curves of In5-In5 and opt10-opt10 synbodies toinfluenza in ELISA.

FIG. 5 shows a synthesized synbody conjugated to Tat peptide.

FIG. 6 shows a hypothetical example of a designed synbody with metalchelator macrocyclic species DOTA.

FIG. 7 shows a synbody including linking to peptide In5 labeled and withAlexa dye for in-vivo imaging to study the bio-distribution of thechemical entity.

FIG. 8 shows a method for improving physiochemical stability of synbodycandidates using hydrolysis.

FIG. 9 shows a graphical representation of peptide binding to influenza(1.5×1010 vp/slide) divided by peptide binding to primary antibody.

FIG. 10 shows a graphical representation of fluorescent intensity versusvirus concentration for candidate peptides with influenza/antibodybinding >4.5.

FIG. 11 shows a graphical representation of Influenza binding tocandidate peptides immobilized on a microarray.

FIG. 12 shows HPLC chromatograms of A) scaffold Sc1, B) opt10 peptide,C) reaction mixture at two hours; and D) purified synbody,opt10-opt10-Sc1.

FIG. 13 shows HPLC chromatograms of A) peptide 5 (P5), B) scaffold Sc1,C) reaction mixture at two hours, D) purified P5-P5-Sc1 synbody I, andE) purified P5-P5-Sc1 synbody II.

FIG. 14 shows HPLC chromatograms of A) opt10 peptide, B) scaffold Sc2,C) reaction mixture at 2 hours, and D) purified synbody opt10-opt10-Sc2.

FIG. 15 illustrates a scatter plot of IAV binding (x-axis) versusantibody only binding (y-axis) to 10,000 peptide microarray.

FIG. 16 graphically illustrates a cytopathic effect assay for MDCK cellstreated with synbody +A/PR/8/34.

FIG. 17A shows reduction of A/PR/8/34 replication as measured by NApositive cells.

FIG. 17B shows reduction of A/PR/8/34 replication as measured by NPpositive cells.

FIG. 18A illustrates Pull-down of NP from viral lysates using P5-P5-Sc2synbody, where the synbody was added to either A/PR/8/34 orA/Sydney/5/97 lysates and captured NP was detected by anti-NP antibody.

FIG. 18B illustrates percent of NP recovered by the synbody for eachlysate.

FIG. 19A is a Western blot of NP pulled down from samples of purified NPusing P5-P5-Sc2.

FIG. 19B shows the determination of binding affinity by pull-down of NPpulled down in FIG. 19A.

FIG. 19C shows SPR sensorgrams of 0.5 and 1.0 nM NP binding toP5-P5-Sc2.

In the drawings, identical reference numbers identify similar elementsor components. The sizes and relative positions of elements in thedrawings are not necessarily drawn to scale. For example, the shapes ofvarious elements and angles are not drawn to scale, and some of theseelements are arbitrarily enlarged and positioned to improve drawinglegibility. Further, the particular shapes of the elements as drawn, arenot intended to convey any information regarding the actual shape of theparticular elements, and have been solely selected for ease ofrecognition in the drawings.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure describes the discovery of peptide affinity reagents(synbodies) for influenza virus. Several features of compositions,methods and systems in accordance with example embodiments are set forthand described in the Figures. It will be appreciated that methods andsystems in accordance with other example embodiments can includeadditional procedures or features different than those shown in theFigures. Example embodiments are described herein with respect to anaffinity reagent for influenza viruses. However, it will be understoodthat these examples are for the purpose of illustrating the principles,and that the invention is not so limited. Additionally, methods andsystems in accordance with several example embodiments may not includeall of the features shown in the Figures.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense that is as “including, but not limited to.”

Reference throughout this specification to “one example” or “an exampleembodiment,” “one embodiment,” “an embodiment” or combinations and/orvariations of these terms means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present disclosure. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Definitions

A synbody is a synthetic entity having at least three components, two ofwhich are compounds having affinity for the same target molecule albeitat different sites within the target molecule and the third being alinker connecting the compounds. The molecular weight of a synbody isusually 500-10,000 kDa and sometimes between about 4 and 5 kDa.

A linker indicates a moiety or group of moieties that connects two ormore discrete compounds in a synbody. A linker is typically bifunctional(i.e., the linker contains a functional group at each end that isreactive with groups located on the compounds to be attached). Linkersinclude amino acids, polypeptides, nucleic acids, small molecules,polymers and particles. Linkers can be linear or branched. Particlesserving as linkers or linkers attached to multiple copies of thecompounds forming a synbody are sometimes referred to as scaffolds.

A spacer is a molecule optionally present between a linker and acompound attached to the linker. A spacer can be, for example, one ormore amino acids or a small organic structure conjugating the linker toa compound.

In some synbodies, the demarcation of compounds, linker and spacer(s) ifpresent is readily apparent, because each has a contiguous or regularlyrepeating structure distinct from another, or because of conjugationchemistries indicating the points of demarcation. However, a preciseunderstanding of demarcation between these components is not usuallynecessary for use.

An isolated peptide or other moiety means that the moiety if found innature is separated at least in part from the molecules with which it isnaturally associated including flanking sequences if the peptide is partof a longer protein. If the peptide or moiety is synthetic, isolatedmeans separated at least in part from chemicals used in its production.An isolated peptide does not exclude the presence of heterologouscomponents, such as a linker, second peptide or pharmaceuticalexcipients not naturally associated with the peptide or used in itssynthesis. An isolated moiety can also be pure (e.g., at least 50, 75,90 or 99% w/w pure) of contaminants

Unnatural amino acids are amino acids other than the twenty naturallyoccurring amino acids that are the building blocks for all proteins, butare nonetheless capable of being biologically or chemically engineeredsuch that they are incorporated into proteins. Unnatural amino acidsinclude D-amino acids, β amino acids, and various other “designer” aminoacids (e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methylamino acids). Synthetic amino acids include ornithine for lysine, andnorleucine for leucine or isoleucine. Hundreds of different amino acidanalogs are commercially available from e.g., PepTech Corp., Mass. Ingeneral, unnatural amino acids have the same basic chemical structure asa naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group. Methods ofmaking and introducing a non-naturally-occurring amino acid into aprotein are known. See, e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647.

Derivatives should have a stabilized electronic configuration andmolecular conformation that allows key functional groups to be presentedto the target binding sites in substantially the same way as the leadmultimer. Identification of derivatives can be performed through use oftechniques known in the area of drug design. Such techniques includeself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis. Computer programs forimplementing these techniques are readily available. See Rein et al.,Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss,N.Y., 1989). Derivatives may have higher binding affinity, smaller size,and/or improved stability relative to a lead multimer. Modifications caninclude N terminus modification, C terminus modification, peptide bondmodification, including, CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂,S═C═NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference.

Specific binding refers to the binding of a compound to a target (e.g.,a component of a sample) that is detectably higher in magnitude anddistinguishable from non-specific binding occurring to at least oneunrelated target. Specific binding can be the result of formation ofbonds between particular functional groups or particular spatial fit(e.g., lock and key type) whereas nonspecific binding is usually theresult of van der Waals forces. Specific binding does not however implythat a compound binds one and only one target. Thus, a compound can andoften does show specific binding of different strengths to severaldifferent targets and only nonspecific binding to other targets.Preferably, different degrees of specific binding can be distinguishedfrom one another as can specific binding from nonspecific binding. Thepeptides and synbodies of the invention show specific binding toinfluenza viruses. Specific binding of synbodies of the inventionusually involves an association constant of 10⁷, 10⁸ or 10⁹ M⁻¹ orhigher.

Abbreviations

Enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodiesand color change to identify a substance.

“Human Influenza A Virus” is abbreviated “IAV.”

“Influenza virus nucleoprotein” is abbreviated “NP.”

“Neuraminidase” is abbreviated “NA.”

“XTT” is an abbreviation for tetrazolium dye which is commerciallyavailable for use in assay protocols including cytotoxicity, andapoptosis assays

N,N-Dimethylformamide (DMF), Trifluoroacetic acid (TFA), Acetonitrile(AcCN), Methyl alcohol (MeOH), Dichloromethane (DCM),1-Hydroxybenzotriazole (HOBt),2-(1-H-benzotroazole-1-yl)-1,3,3-tetramethyluronium Hexafluorophosphate(HBTU), Nmethylmorpholine (NMM), Trifluoroacetic acid (TFA),N,N-Diisopropylethylamine (DIPEA),Triisopropylsilane (TIPS),3,6-Dioxa-1,8-octane-dithiol (DoDt),1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl (ivDde),Fluorenylmethoxycarbonyl (Fmoc), Kaiser reagents (1. Ninhydrinesolution; 6% in ethanol, 2. Potassium cynide in pyridine, 3. Phenol in80% ethanol) Carboxymethyldextran (CMD). All the regents were used asprovided by vendor unless specified.

It has been successfully demonstrated that high affinity ligands (orsynbodies) to any given target protein can be prepared by linking lowaffinity 15-20 AA long peptides. The present disclosure demonstrates theextension of this simple, robust platform technology for rapidproduction of high affinity ligands for viruses. To explore this concepton viruses, one of the laboratory strains - Influenza A/PR/8/34 wasselected.

Referring now to FIG. 1, there shown is an overview of an example ofhigh affinity synbody development process. In one example, a targetvirus was screened over a small library of 10,000 random peptides(CIM10K) to identify lead peptides with moderate affinity for a targetvirus and constrained on a scaffold is shown. As described herein,simple bivalent linking can produce a 10 to 100-fold improvement inbinding affinity over the starting peptides and this should convert twopeptides with modest binding affinity into a high affinity ligandwithout having prior knowledge of the binding sites and can extend toviruses and thus help in making high affinity ligands without priorknowledge of virus genetics.

A peptide microarray platform for the selection of peptide candidatesfor any given target including but not limited to proteins, pathogen,cells, and antibodies was established. A library of 10,000 randomsequences of 20 mer peptides containing completely random 17 AApositions and three amino acids (G, S, C) linker at the N-terminus wereimmobilized in array format. Cysteine at N-terminus was used for surfaceimmobilization through reactive sulfhydryl group and has addedadvantages including, (a) only complete synthesized peptide sequenceswill be immobilized on the surface, and (b) increased protease stabilityof the synbody due to amide bond at the N-terminus. Virus particles (10⁸virus/slide) were screened on a microarray and antibody based detectionmethodology was used in screening. Both primary and secondary antibodieswere screened as controls to remove false positives in the selectionprocess. Selected peptides as listed in Table 1 were validated in ELISAbinding assay before multivalent affinity ligand (synbody) synthesis.

TABLE 1 List of Influenza Binding Peptides SEQ ID NO: Peptide NamePeptide Sequence  1 P-5 (In5) CSGDMYEYNPFQGNHIYNKK 16 P-75CSGDSPMGYYHQKTSPWADK 17 P-204 CSGQFSAKKYWEIKPMDYWK 18 P-210CSGQYSQQSSSYGQQMFKKE 19 P-54 CSGAQEWAAKSYKWNKDGYL 20 P-68CSGDMHWGYQDGKTLVPTSK 21 P-71 CSGDPTHATEPKRYEAYNDH 22 P-88CSGEMWAIMPPIIKPDNKGH 23 P-125 CSGHNIYAQYGYPYDHMYEG 24 P-134CSGKDHNAQDQESVHWKYKG 25 P-147 CSGKRYLQKGKGALRGLYIF 26 P-149CSGKSQEIGDPDDIWNQMKW 27 P-150 CSGKTEHYMPNNNTFGYEYE 28 P-157CSGLLHELPDDYEKINPQKY 29 P-158 CSGLLYHFKVGLRTMKISMM 30 P-166CSGMKQPKHNKINDNPKAYE 31 P-174 CSGNETAPDNTYRYKQSAQK 32 P-227CSGSFNQEYFPYPMIDYLKK 33 P-241 CSGTANELLYYKNYGVKNPK 34 P-243CSGTEEKYINDSNFADEKGH

With an aim to develop ligands with high affinity influenza, oneobjective was to improve lead peptides affinity for Influenza A/PR8/34.We selected one of the lowest binding peptides (P5) for binding affinityimprovement. one of the lowest binding peptide (In5) was selected forbinding affinity improvement. Due to the linear nature of most of the 20AA peptides, it is possible to make single AA substitutions to identifyAA's that increase affinity. These identified AA substitutions wereadded in combination of 5-8 AA acids to design a new peptide sequencewith possible improved binding. Taking advantage of this platform,D-amino acids substitutions were used to increase protease stability indownstream applications. For this, each amino acid in the lead peptidesequence was replaced with 19 D-AA's (a, r, n, d, e, q, g, h, i, l, k,m, f, p, s, t, w, y, v) to design an amino acid point variant peptidelibrary containing 324 peptides for the selected lead peptide (P5).Microarrays with 324 peptides were screened and analyzed similar toCIM10k microarrays. A heat map was generated by calculating relativevalves with respect to lead P5 which clearly showed the effect of eachAA substitution in the lead sequence. A single amino acid substitutionwith arginine or proline in place of glutamic acid alone gave 40-60 foldimprovements in binding over original sequence. Also, argininesubstitution at aspartic acid or asparagine position improved affinity20-60 fold. On the basis of these results a plurality of new optimizedpeptides, listed in Table 2, were designed and synthesized by additionof 5-6 amino acid combinations using arginine, proline, and lysine.These new optimized peptides were synthesized and directly used forsynbody construction on a bivalent scaffold.

TABLE 2 List of designed mutant peptides for peptide (In5) SEQ ID NO:Peptide Name Peptide Sequence  1 P-5 (In5) CSGDMYEYNPFQGNHIYNKK  2 Opt1CSGDMYrYNPFQGNHIYNKK  3 Opt2 CSGrMYEYNPFQGNHIYNKK  4 Opt3CSGDMYpYNPFQGNHIYNKK  5 Opt4 CSGrMYpYNPFQGNHIYNKK  6 Opt5CSGrMYrYNPFQGNHIYNKK  7 Opt6 CSGDMYEYNPFQGNHIYrKK  8 Opt7CSGrMYpYNPFQGNHIYrKK  9 Opt8 CSGtMYEYNPFQGNHIYNKK 10 Opt9CSGDMYEYrPFQGNHIYNKK 11 Opt10 CSGrMYpYrPFQGNHIYrKK 12 Opt11CSGtMYpYrPFQGNHIYrKK 13 Opt12 CSGrMYpYrPFQrkHIYrKK 14 Opt13CCSGDrYEYNPFQGkHIYNK 15 Opt14 CSGrMYEYNPFQGkHIYrKK

For synbody construction, two bivalent peptide based scaffolds “Scaffold1 and Scaffold 2” (as shown in FIG. 2A and FIG. 2B) were designed withmaleimide groups and sulfhydryl conjugation chemistry was chosen tomimic the orientation and chemistry of the peptides on the microarrays.Both the scaffolds and peptides were prepared via standard solid phasepeptide chemistry (SPPS). Therefore modifications can be made accordingto desired applications. Also, glutamic acid and 6 AA sequence (GSGKSG(SEQ ID NO: 35)) were added to increase solubility as well as for easydetection on MALDI-TOF for characterization and quality controlpurposes. The addition of an inert orthogonal group “propargyl glycine”in the scaffold-1 added an extra coupling site for the development ofvarious downstream assays. To test the modularity of thepeptide-scaffold linking, designed another scaffold with a peptideIMKPFETHRLGPERFDGSC (SEQ ID NO: 36) attached at the C-terminus and twounits of mini-PEG molecule to increase solubility.

The synbodies were synthesized by conjugating peptides with cysteine atN-terminus to maleimide functionalized scaffold at pH 7.0 in presence ofTEA. This way amide bond at the C-terminus of the peptide becameN-terminus and could be less susceptible to protease cleavage and one ofthe reason of designing peptide libraries with N-terminus cysteine.Reaction of each peptide with the scaffold results only homo-bivalentsynbodies. The synthesized Synbodies were characterized by MALDI-TOF andpurified by HPLC for functional assays. The sulfhydryl assisted covalentattachment of cysteine containing peptides to scaffolds is very rapidand scalable with high yields for the fast production of large number ofligands in very short time. In the last 10 years, technical advances inpeptides production and scale have increased the economics and eminenceof peptides in drug development; making this platform more costeffective comparable to existing ligand discovery and developmentsystems. The homo-bivalent synbody candidates (P-5) prepared onscaffold-1 were screened unpurified and compared for their bindingbehavior against target virus via surface plasmon resonance (SPR). Fourout of fifteen synbodies showed improved binding (Table 2) andidentified synbody opt10-opt10 as lead candidate with >100 foldimprovement over wild type (WT) (P5-P5) candidate.

TABLE 3 Synbodies constructed and tested for influenza binding by SPRSEQ ID NO: Synbody Name Fold Change versus WT Synbody WT 1 2-2 opt1-opt16 3-3 opt2-opt2 3 4-4 opt3-opt3 n.b. 5-5 opt4-opt4 10 6-6 opt5-opt5 n.b.7-7 opt6-opt6 4 8-8 opt7-opt7 45 9-9 opt8-opt8 1 10-10 opt9-opt9 1 11-11opt10-opt10 131 12-12 opt11-opt11 47 13-13 opt12-opt12 n.b. 14-14opt13-opt13 n.b. 15-15 opt14-opt14 107 n.b.—no binding

To further ensure the results, purified optimized (opt10-opt10-Sc-1) andwild type (P5-P5-Sc-1) synbody candidates tested along with optimized(opt10) and selected lead (In5) peptides in ELISA binding assay. Theoptimized peptide showed approximately 1000 fold improvement overidentified lead peptide which on linking on either of the scaffoldsshowed 40 times increment in binding affinity compared to singleoptimized peptide alone. Overall, in just two steps a 24,000 foldimproved optimize synbody was designed with binding affinities (KD=1nM)which is in range of anti-influenza NA monoclonal antibody (FIG. 5).These results demonstrated that attachment of extra groups/peptides doesnot affect the binding constant and the modularity of the synbody designthat it can be modified for any new application without compromising thebinding affinities.

It is possible that these synbodies could interfere with an influenzainfection, therefore, we designed several modifications to the synbodyscaffold that can improve the performance or aid in the in vivocharacterization of the synbody. It is possible that synbodies caninterfere with virus replication inside the cells, therefore we designeda scaffold with a function group whereby it is easy to attach a cellpenetrating peptide, such as Tat (illustrated in FIG. 5) to the synbody.It is important to understand the bio-distribution of a potentialtherapeutic, therefore we designed synbodies with a metal chelatormacrocyclic species like DOTA as shown in FIG. 6 and one with an Alexadye (as shown in FIG. 7) for in-vivo imaging to study thebio-distribution of the chemical entity.

Method to Improve Physiochemical Stability of Synbody Candidates.

Referring now to FIG. 8 a method for improving physiochemical stabilityof synbody candidates using hydrolysis is shown. In this hypotheticalexample, sulfhydryl conjugation chemistry was selected for rapidproduction of synbodies. But in in-vivo functional assays, thismaleimide coupled synbodies undergoes exchange reaction with albumin,glutathione and other cysteine moieties present in the plasma. Thissuccinimide exchange reaction results in new products formed viasulfhydryl conjugation of scaffold to albumin, glutathione and othercysteine moieties present in the plasma. This led to reduced circulatingsynbody amount in the plasma. A method to avoid succinimide exchangereaction in-vivo was developed and for this we have hydrolyzed thesuccinimide ring first before testing in-vivo. This completely inhibitssuccinimide exchange and desired synbody is circulating without anymodifications. This concept has been tested over many synbodiessynthesized using maliemide chemistry with different peptide sequencesand scaffold types and found that succinimide ring opening totallyinhibits sufhydryl exchange and increase circulating synbody half-lifein-vivo.

EXAMPLES AND SUPPORTING METHODS Supporting Method (SM1)—Screening ofInfluenza A PR/8/34 H1N1 on Peptide Microarray

Peptide microarrays were washed with 90% TFA for 5 minutes followed byrinsing with DMF (1×), 95% ethanol (1×), and double-distilled water(ddH2O) (2×). Microarrays were dried by centrifuging at 1500 rpm for 2minutes. After drying, microarrays were blocked with 600 ul blockingbuffer [3% bovine serum albumin (BSA), 0.05% Tween20, 0.134 mg/mlMercaptohexanol in 1× Tris buffered saline (TBS)] in a humidity chamberfor 1 hour at room temperature. After blocking, microarrays were washedwith washing buffer (1×TBS+0.05% Tween20) followed by two washes withddH2O. The following reagent was obtained through the NIH Biodefense andEmerging Infections Research Resources Repository, NIAID, NIH: InfluenzaA Virus, A/Puerto Rico/8/34 (H1N1), NR-348. Influenza solutions (250 uL)at five different concentrations (7.5×10⁸, 1.5×10⁹, 3×10⁹, 7.5×10⁹,1.5×10¹⁰ vp/ml) were incubated in competition with protease inhibitedbovine serum (5 mg/mL) in Agilent microarray chambers for 1 hour at 37°C. Microarrays were washed as before and probed with 5 nM, 250 uL mousemonoclonal anti-influenza NA (BEI Resources, Cat. No: NR-4540) with samecompetitor in Agilent microarray chambers for 1 hour at 37° C.Microarrays were washed and probed with 5 nM, 250 uL AlexaFluor-647conjugated anti-mouse secondary antibody (Life Technologies, Cat. No:A21235) for 1 hour at 37° C. All the slides were washed, dried, andscanned on an Agilent Microarray Scanner. Each sample was run in 4replicates. As a negative control the primary antibody was screened inabsence of pathogen. Data were analyzed with Agilent GeneSpring softwarewithout additional normalization.

To select influenza binding peptide candidates, the following selectioncriteria were used: (a) signal ratio of influenza/antibody only >2 (b)less than 30% deviation between peptide replicates (c) increasing signalwith increasing viral concentration. As unpurified peptides were used inthe 10K peptide library, this initial list of candidates (114) wasfiltered by synthesis quality, based on their MALDI mass spectra, inorder to eliminate any peptides whose binding might be mediated byunremoved protecting groups. A total of 57 peptides passed this step andwere synthesized (Sigma Custom Peptide, The Woodlands, Texas) with anN-terminal biotin tag for use in subsequent assays.

Supporting Method (SM2)—Screening of Influenza Binding Peptides byPeptide Microarray

The selected peptide candidates were printed on polymer slides as in theinitial screening but were printed at three different concentrations(˜0.5 mg/mL, 1 mg/mL, 2 mg/mL) for the secondary screening. This wasdone to test that peptide binding from the initial screening wasreproducible and that influenza binding occurred at other peptidedilutions. This should reduce the chance that the observed peptidebinding was simply a product of avidity of high levels of immobilizedpeptide. In addition, we compared binding between live H1N1 and H1N1inactivated by UV (Advanced Biotechnologies, Inc. #10-273-500) orformalin treatment; inactivated following a standard inactivationprotocol. Data from the three preparations of influenza binding to theimmobilized peptides was very comparable; therefore UV inactivatedinfluenza was used for subsequent assays.

Supporting Method (SM3)—Screening of Influenza Binding Peptides by ELISA

Nunc MaxiSorp flat bottom 96 well ELISA plates (VWR # 62409-002) werecoated with 50 uL of 0.2 ug/well of UV inactivated Influenza A PR/8/1934H1N1 in ELISA coating buffer and kept overnight at 4° C. Plates werewashed with 1×PBST followed by blocking with 100 uL 3% BSA in 1×PBST for2 hours at 37° C. After washing plates, biotin labeled peptides wereadded in a concentration range from 0.39 to 100 uM in dilution buffer[0.1% BSA+1×PBST +0.05% v/v Tween20] and incubated for 1 hour at 37° C.Plates were washed and 100 uL of 1:2000 streptavidin-HRP (ThermoScientific # N100) was added and incubated for 1 hour at 37° C. Plateswere washed and 100uL of 3,3′, 5,5′-tetramethylbenzidine (TMB) (ThermoScientific # N301) was added and incubated in the dark for 15 minutes atroom temperature. The reaction was quenched by the addition of 100 ul of0.5M HCl and read immediately at 450 nm using a microplate reader(Spectra MAX 190, Molecular Devices, Inc.). ELISA dilution buffer wasrun as control on influenza coated wells. Also, two peptideconcentrations 0.39 uM and 100 uM were run on non-influenza coatedwells.

Supporting Method (SM4)—Synthesis of Scaffolds (Sc1 and Sc-2)

All the Fmoc-amino acids and coupling reagents were from AAPPTEC(Louisville, Ky.). FmocPal-PEG resin (0.2 mmole/gram) was purchased fromLife Technologies (Grand Stateley, N.Y.). Maleimidopropionic acid wasfrom Bachem (Torrance, Calif.). Trifluoroacetic acid (TFA), piperidine,N,N-diisopropylethylamine (DIPEA) were purchased from Spectrum (LosAngeles, Calif.). And remaining reagents/solvents were obtained fromFisher Scientific.

S3 Both scaffolds (Sc1 and Sc2) were synthesized via Fmoc based solidphase peptide synthesis. The synthesis was carried out at 0.5 mmolescale on Fmoc-Pal-PEG resin Rink amide resin (0.2 mmole/g). Thesequences are shown in FIG. 3. Following removal of Fmoc-protectinggroup by 20% piperidine in DMF for 5+15 minutes, the stepwise assemblyof the peptide sequence was performed via addition of the appropriateFmoc protected amino acids. The N-terminus was substituted withmaleimide group manually by treating with 5 fold excess ofmaleimidopropionic acid in the presence of 5 fold excess of HOBt anddiisopropylcarbodiimide in DMF. The resin was agitated at roomtemperature for 1 hour followed by standard washings with DMF (3 times,1 minute per wash), MeOH (2 times, 1 minute per wash), DCM (2 times, 1minute per wash), DMF (3 times, 1 minute per wash). An aliquot of resinwas taken after MeOH wash for qualitative Kaiser Test. The finalprotected scaffolds was treated with cleavage cocktail (TFA: H2O: TIPS:90:5:5) for 2 hours at room temperature and precipitated in cold diethylether. The precipitated construct was cooled for 15 minutes in −80° C.refrigerator to ensure complete precipitation. The solid was separatedfrom the diethyl ether by centrifugation and the top phase was decantedoff and pellet re-suspended with another addition of dry diethyl ether.The cooling and centrifugation process was done in triplicate. Uponcompletion, the construct was dried and dissolved in water for HPLCpurification. The construct was purified on reverse-phase HPLC (Agilent1200 Series HPLC) using a Phenomenex Luna 5u semi-preparative (10 mm×250mm) C-18 column using solvent system A: 0.1% TFA in H2O; solvent B: 90%CH3CN in 0.1% TFA with a linear gradient method, (0 min, 10% B; 2 min,10% B; 20 min, 45% B; 25 min, 95% B; 27 min, 95% B; 30 min, 100% B; 33min, 10% B) with flow rate of 4 mL/min at a wavelength of 280 nm. Theappropriate fractions were collected and analyzed by MALDI-TOF massspectrometry. The correct fraction was then lyophilized. These scaffoldswere then purchased at large scale (>90% purity) from Sigma CustomPeptide and used for subsequent synbody preparation.

Supporting Method (SM5)—Conjugation of Synbodies

General Method—In the synthesis of a synbody, the peptide and thescaffold are separately dissolved in 30% acetonitrile in water. Oneequivalent of the scaffold is first mixed with two equivalents of thepeptide; the pH of the reaction mixture is then adjusted to 6.5-7.0 withthe addition of dilute triethylamine (TEA) (10% in acetonitrile). Thereaction is allowed to proceed at room temperature without shaking andmonitored by HPLC and MALDI-MS. The yield of the reaction is determinedby integration of the peaks assigned to the desired product.

Example 1

Synbody opt10-opt10-Sc1. HPLC analysis of the reaction mixture showedthat the scaffold, Sc1, was completely consumed in two hours (FIG. 12).A single peak at 15.02 min corresponded to the desired synbody product.The yield of the desired synbody was estimated as >90% by theintegration of the peak. MALDI-MS: calculated for M+H 6570.163(monoisotopic), found 6572.685. Referring now to FIG. 13 HPLCchromatograms of A) scaffold Sc1, B) opt10 peptide, C) reaction mixtureat two hours; and D) purified synbody, opt10-opt10-Sc1 is shown.

Example 2

Synbody opt10-opt10-Sc2. HPLC analysis showed that, after two hours ofreaction, the scaffold, Sc2, was completely consumed (FIG. 13). The peakat 16.08 min is characterized by MALDI-MS as the desired synbodyproduct, the yield of the desired product was estimated as >90% by HPLCintegration. MALDI-MS: calculated for M+H, 8818.239 (monoisotopic),found 8819.824. Referring now to FIG. 14 HPLC chromatograms of A)peptide 5 (P5), B) scaffold Sc1, C) reaction mixture at two hours, D)purified P5-P5-Sc1 synbody I, and E) purified P5-P5-Sc1 synbody II areshown.

Example 3

Synbody P5-P5-Sc1. HPLC analysis of the reaction showed that thescaffold Sc1, was completely consumed in two hours. However, there weretwo peaks (t=16.9 min and t=17.34 min) corresponding to the desiredproduct (t=17.34 min), while the peak at 16.52 contains both the monoconjugation of peptide 5 (P5) to Sc1 and the P5-P5-Sc1 synbody. Thecombined yield of synbody I and II, is determined as 73% based HPLCintegration. MALDI-MS: calculated for M+H 6383.731 (monoisotopic), found6385.485 (synbody I), 6385.096 (synbody II). FIG. 4 shows binding curvesof In5-In5 and opt10-opt10 synbodies to influenza in ELISA. Affinityligand concentration is plotted versus A450 (y-axis).

Supporting method (SM6)—SPR Screening of Synbodies

Determination of the binding of synbodies to immobilized influenza viruswas performed on a Biacore 4000. The series S sensor chip CM5, aminecoupling reagents and HBS-EP were obtained from GE Healthcare. An amineimmobilization protocol was performed at 25° C. using 10 mM NaHCO3, pH5.0 as the immobilization buffer. All five spots on one flow cell of theCM-5 chip were activated by a 10 minute injection of a freshly prepared1:1 solution of 400 mM EDC: 100 mM NHS in water. Spots 1, 2, 4 and 5were treated with a solution of UV inactivated Influenza A PR8/1934 (50μg/ml) in 10 mM NaHCO3, pH 5.0 for 8, 14, 16, and 10 minutes at a flowrate of 10 mL/min. Any residual active sites were then quenched by a 5minute pulse of ethanolamine (1M, pH 8.5). Synbody samples at 100 uMwere injected at a flow rate of 30 uL/min over the flow cell surface.Buffer injections identical to the analyte were included throughout theanalysis for the purpose of double referencing. The surfaces wereregenerated with one 30-s injection of pH 3.0 glycine. The binding levelfor each injection was determined using Biacore 4000 EvaluationSoftware.

Supporting Method (SM7)—ELISA Binding Assay for Synbodies

Nunc MaxiSorp flat bottom 96 well ELISA plates (VWR # 62409-002) werecoated with 100 uL of lug per well of UV inactivated Influenza APR/8/1934 H1N1 in standard ELISA coating buffer and kept overnight at 4°C. Plates were washed with 1x PBST followed by blocking with 200 uL 6%BSA (ELISA grade fraction V) in 1×PBST for 2 hours at room temperature.Plates were washed twice with 1×PBST and biotin labeled synbodies wereadded in ELISA dilution buffer (1% BSA +1×PBS+0.05% v/v Tween20) andincubated for 1 hour at room temperature. After washing, 100 uL of1:100,000 streptavidin-HRP was added and incubated for 1 hour at roomtemperature. Plates were washed and 100 uL of TMB was added. Plates wereincubated in the dark for 8 minutes at room temperature. The reactionwas quenched by addition of 100 uL of 0.5 M HCl and read immediately at450 nm using micro plate reader. ELISA dilution buffer was run as acontrol on influenza coated wells. Anti-influenza NA (BEI Resources,Cat. No: NR-4540) antibody (5 nM) and unrelated synbody was used aspositive and negative control respectively.

Referring now to FIG. 9 a graphical representation of peptide binding toinfluenza (1.5×1010 vp/slide) divided by peptide binding to primaryantibody is shown. Median fluorescent intensity was used for eachcondition.

Referring now to FIG. 10 a graphical representation of fluorescentintensity versus virus concentration for candidate peptides withinfluenza/antibody binding >4.5 is shown.

Referring now to FIG. 11 a graphical representation of Influenza bindingto candidate peptides immobilized on a microarray is shown.

Referring now to FIG. 14 HPLC chromatograms of A) opt10 peptide, B)scaffold Sc2, C) reaction mixture at 2 hours, and D) purified synbodyopt10-opt10-Sc2 are shown.

Having described properties of synbodies hereinabove, furtherdescriptions of other useful properties of synbodies are describedhereinbelow to promote understanding of the advantages and uses of themethods, agents and techniques disclosed herein. In particular it hasbeen found that synbodies inhibit the cytopathic effect of influenza onMDCK cells, that synbodies inhibit influenza replication in MDCK cells,and that the P5-P5 synbody, in particular, binds nucleoprotein.

Referring now jointly to FIG. 15 and FIG. 16, FIG. 15 shows a scatterplot of IAV binding (x-axis) versus antibody only binding (y-axis) to10,000 peptide microarray is illustrated, and FIG. 16 graphicallyillustrates a cytopathic effect assay for MDCK cells treated withsynbody +A/PR/8/34. The illustrated examples support the finding thatsynbodies inhibit the cytopathic effect of influenza on MDCK cells.Influenza binding peptides were conjugated to a bivalent scaffold (Sc1,as best shown in FIG. 2A) and screened for viral inhibition in an invitro inhibition assay [3] with A/PR/8/34 H1N1 infected Madin-DarbyCanine Kidney (MDCK) cells. A dimeric synbody (FIG. 16—open circles)composed of 2 copies of the same binding peptide (5-5-Sc1) was found toinhibit virus induced cell death (as measured by XTT assay) with an IC₅₀of ˜2.5 μM.

Referring now jointly to FIG. 17A and FIG. 17B, FIG. 17A shows reductionof A/PR/8/34 replication as measured by NA positive cells, and FIG. 17Bshows reduction of A/PR/8/34 replication as measured by NP positivecells. The synbody (dot) was compared to a 1:1,000 dilution of sera frommice immunized with A/PR/8/1934 (triangle). *p<0.05, **p<0.01. Theillustrated examples support the finding that synbodies inhibitinfluenza replication in MDCK cells. To directly measure inhibition ofA/PR/8/34 replication, we tested P5-P5-Sc2 inhibitory activity in aplaque reduction assay adapted from [4]. In this assay, P5-P5-Sc2 andA/PR/8/34 were added to MDCK cells at a multiplicity of infection of0.01 for 2 hours before a layer of Avicel® microcrystalline cellulosewas added. (Avicel® is a microcrystalline cellulose product commerciallyavailable from FMC BioPolymer 1735 Market Street Philadelphia, Pa.19103.) Cells were incubated for 24 hours, fixed, permeabilized andinfected cells were detected with either an anti-neuraminidase (NA) oranti-NP monoclonal antibody. Influenza positive plaques were counted andplaque inhibition was plotted as a function of synbody concentration (asbest seen in FIG. 17B). Sera from mice previously immunized withA/PR/8/34 was used as a positive control and showed virtually completeinhibition of virus replication. The synbody inhibited virus replicationwith an IC₅₀˜2.5 μM, in good agreement with the cytopathic effect assaydescribed above with reference to FIG. 16B, although inhibition did notfit to a classical inhibition model (black line). The synbody was thentested against two additional influenza strains, A/CA/7/2009 H1N1 andA/Sydney/5/1997 H3N2, and had similar inhibition constants despite thedifferences in each strain (Table 4).

TABLE 4 Viral yield reduction as measured by NP positive cells. IC₅₀A/PR/8/34 H1N1 2.5 μM A/CA/7/09 H1N1 2.5 μM A/Sydney/5/97 H3N2 2.5 μM

Referring now jointly to FIG. 18A and FIG. 18B, FIG. 18A illustratesPull-down of NP from viral lysates using P5-P5-Sc2 synbody, where thesynbody was added to either A/PR/8/34 or A/Sydney/5/97 lysates andcaptured NP was detected by anti-NP antibody, and FIG. 18B illustratespercent of NP recovered by the synbody for each lysate. These examplessupport the finding that the P5-P5 synbody binds nucleoprotein. As thesynbody was developed using the intact influenza virus, we believed thathemagglutinin (HA) was the likely target. However, P5-P5-Sc2 did notinhibit hemagglutination of A/PR/8/34. This led us to investigate whichviral protein the synbody bound. We tested P5-P5-Sc2 in a pull-downassay against A/PR/8/34 H1N1 and A/Sydney/5/1997 H3N2 viral lysate. AsNP is one of the most abundant viral proteins, we probed the membranewith an anti-NP antibody and found that the synbody pulled-down NP fromboth lysates (FIG. 18). NP is highly conserved amongst influenza strainsand is 100% conserved between these two strains. As seen in FIG. 18B,the percent of NP recovered from each lysate is roughly the same.

Referring now jointly to FIG. 19A, FIG. 19B and FIG. 19C, we then soughtto estimate the binding affinity of P5-P5-Sc2 for NP. As shown in theWestern plot of FIG. 19A the synbody was used to pull down NP fromincreasing concentration solutions of purified NP. As shown in FIG. 19B,recovered NP was quantified and the KD was determined to be 62±26 nM. ASillustrated in FIG. 19 C, SPR sensorgrams of 0.5 and 1.0 nM NP bindingto P5-P5-Sc2As. NP forms oligomers at higher concentrations, we screened0.5 and 1.0 nM solutions of purified NP by SPR against immobilizedP5-P5-Sc2 and estimated the KD at ˜30 nM (FIG. 19C). Low concentrationswere used to ensure NP existed as a monomer in solution.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the invention. Unlessotherwise apparent from the context any step, element, embodiment,feature or aspect of the invention can be used with any other. Allpublications (including GenBank or Swiss-Prot Accession numbers and thelike), patents and patent applications cited are herein incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent and patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. If more than one version of a sequenceis associated with a deposit number at different times, the versionassociated with the deposit number at the time of filing the applicationis meant.

REFERENCES

1. Jonges, M.; Liu, W. M.; van der Vries, E.; Jacobi, R.; Pronk, I.;Boog, C.; Koopmans, M.; Meijer, A.; Soethout, E. Journal of ClinicalMicrobiology 2010, 48, 928.

2. Tarus, B.; Chevalier, C.; Richard, C.-A.; Delmas, B.; Di Primo, C.;Slama-Schwok, A. Plos One 2012, 7, e30038.

3. Grund S, Adams O, Wählisch S, Schweiger B (2011) Comparison ofhemagglutination inhibition assay, an ELISA-based micro-neutralizationassay and colorimetric microneutralization assay to detect antibodyresponses to vaccination against influenza A H1N1 2009 virus. Journal ofVirological Methods 171: 369-373.

4. Matrosovich M, Matrosovich T, Garten W, Klenk H D (2006) Newlow-viscosity overlay medium for viral plaque assays. Virol J 3: 63.

What is claimed is:
 1. An agent comprising a first peptide having anamino acid sequence comprising a first mutant of SEQ. ID NO: 1 and asecond mutant of SEQ ID NO: 1, wherein the first and second mutants arelinked and consist of linked mutant peptides SEQ ID NOs: 2-2, 3-3,5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15.
 2. The agent of claim 1wherein the first and second mutant peptides are linked to a scaffoldstructure to synthesize a composition having an affinity for a targetmolecule.
 3. The agent as in claim 1 or 2 wherein the synthesizedcomposition has an affinity for influenza viruses.
 4. The agent as inclaim 2 or 3 wherein the scaffold structure has the structure:


5. The agent as in claim 2 or 3 wherein the scaffold structure has thestructure:


6. A composition comprising or consisting of SEQ ID NO: 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or
 15. 7. A composition comprising orconsisting of the structure

where In5 comprises or consists of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or
 15. 8. A method of detecting an influenza virus,comprising contacting a sample suspected of containing influenza viruswith an agent of claim 1, and measuring binding of the agent to thesample compared with a control lacking influenza virus, an increase inbinding relative to the control providing an indication of presence ofinfluenza virus.
 9. The method of claim 8, wherein the sample is from apatient.
 10. The method of claim 9, wherein the agent further comprisesa therapeutic molecule linked to the agent.
 11. A method of diagnosing apatient for an influenza virus, comprising contacting a sample suspectedof containing influenza virus with an agent of claim 1, and measuringbinding of the agent to the sample compared with a control lackinginfluenza virus, an increase in binding relative to the controlproviding an indication of presence of influenza virus.
 12. The methodof claim 11, wherein the agent further comprises a therapeutic moleculelinked to the agent.
 13. A method of detecting an influenza virus,comprising contacting a sample suspected of containing influenza viruswith a composition of claim 7, and measuring binding of the compositionto the sample compared with a control lacking influenza virus, anincrease in binding relative to the control providing an indication ofpresence of influenza virus.
 14. The method of claim 13, wherein thesample is from a patient.
 15. The method of claim 14, wherein the agentfurther comprises a therapeutic molecule linked to the agent.
 16. Amethod of diagnosing a patient for an influenza virus, comprisingcontacting a sample suspected of containing influenza virus with acomposition of claim 7, and measuring binding of the composition to thesample compared with a control lacking influenza virus, an increase inbinding relative to the control providing an indication of presence ofinfluenza virus.
 17. The method of claim 16, wherein the agent furthercomprises a therapeutic molecule linked to the agent.
 18. A diagnosticagent for influenza comprising a first peptide having an amino acidsequence comprising a first mutant of SEQ. ID NO: 1 and a second mutantof SEQ ID NO: 1, wherein the first and second mutants are linked andconsist of linked mutant peptides SEQ ID NOs: 2-2, 3-3,5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15; wherein the first andsecond mutant peptides are linked to a scaffold structure to synthesizea composition having an affinity for influenza viruses; and whereincontacting a sample suspected of containing influenza virus with thesynthesized composition, and measuring binding of the synthesizedcomposition to the sample compared with a control lacking influenzavirus, an increase in binding relative to the control providing anindication of presence of influenza virus.
 19. A therapeutic agent forinfluenza comprising a first peptide having an amino acid sequencecomprising a first mutant of SEQ. ID NO: 1 and a second mutant of SEQ IDNO: 1, wherein the first and second mutants are linked and consist oflinked mutant peptides SEQ ID NOs: 2-2, 3-3,5-5,7-7,8-8,9-9,10-10,11-11,12-12, or 15-15; wherein the first andsecond mutant peptides are linked to a scaffold structure to synthesizea composition having an affinity for influenza viruses; and wherein theagent further comprises a therapeutic molecule linked to the synthesizedcomposition.
 20. A method for inhibiting the cytopathic effect ofinfluenza on MDCK cells comprising: conjugating influenza bindingsynthetic peptides to a bivalent scaffold; screening for viralinhibition in an in vitro inhibition assay using A/PR/8/34 H1N1 infectedMadin-Darby Canine Kidney (MDCK) cells; and using a dimeric synbodycomposed of 2 copies of the same binding peptide to inhibit virusinduced cell death with an IC₅₀ of ˜2.5 μM.
 21. A method for inhibitinginfluenza replication in MDCK cells comprising: conjugating peptideswith cysteine at N-terminus to a maleimide functionalized scaffold at pH7.0 in presence of dilute triethylamine to create a synbody; adding thesynbody and A/PR/8/34 H1N1 infected Madin-Darby Canine Kidney (MDCK)cells at a multiplicity of infection of at least 0.01; then adding alayer of microcrystalline cellulose; and incubating the MDCK cells. 22.The of claim 21 wherein the sybody consists of a P5-P5-Sc2 synbody.